The increasing use of exotic and hybrid poplar species in forestry and the lack of genetic barriers between most poplar species may present a risk to the genetic integrity of native poplar species. To monitor any spontaneous hybridization and (or) introgression from exotics into native species, it is essential to have a system for the quick and reliable identification of species. We developed a set of single nucleotide polymorphism (SNP) markers that allows the distinction between five commercially important species of poplar ( Populus balsamifera L., Populus deltoides Marsh., Populus trichocarpa Toor. ex Gray, Populus nigra L., and Populus maximowiczii Henry) and their hybrids. Six genomic regions spanning 6.1 kb were screened at the DNA sequence level to search for discriminating SNPs among the five species. A total of 245 SNPs and indels were found, 86 of which were species specific. A subset of 12 species-specific SNPs was chosen for use with high-throughput SNPstream technology. In addition, 32 species-specific SNPs and indels were found that can be assayed using restriction enzymes. Application of the developed markers to a set of hybrid clones showed that the markers are not only useful for monitoring introgression but also for the verification of breeding material.